Method for assaying lower respiratory tract infection or inflammation

ABSTRACT

The present invention relates to a reliable method of prediction of lower respiratory tract infection or inflammation in humans, wherein the level of pancreatic stone protein/regenerating protein (PSP/reg) is determined in serum, and a high level is indicative of the development and the severity of the disease, allowing the classification of patients according to risk.

FIELD OF THE INVENTION

The present invention relates to a method of prognosis or survivalprediction and/or diagnosis of infections or inflammatory diseases ofthe airways and lungs, in particular for prognosis of the development ofacute exacerbation of chronic obstructive pulmonary disease (AECOPD) orventilator associated pneumonia (VAP). The invention allows for thedetermination of the level of pancreatic stone protein/regeneratingprotein (PSP/reg) in body fluid samples of a patient and sortingpatients according to risk.

BACKGROUND OF THE INVENTION

Lower respiratory tract infections, i.e. acute bronchitis, acuteexacerbations of chronic obstructive pulmonary disease (COPD) orpneumonia, account for almost 10% of the worldwide burden of morbidityand mortality. Detecting the presence, defining the cause, andpredicting the severity of lower respiratory tract infections areconstant challenges for the treating clinician. Among the most commonlyused biomarkers for the detection and management of lower respiratorytract infections are leukocyte counts, C-reactive protein, andprocalcitonin. Unfortunately, none of these biomarkers is completelysatisfying.

Pancreatic stone protein/regenerating protein (PSP/reg) belongs to afamily of lectin-binding proteins that were identified initially inpatients with pancreatitis (L. Multigner et al., Gastroenterology 1985;89:387-391). PSP/reg has been studied predominantly in the pancreas.Under conditions of acute or chronic pancreatitis, it is highlyup-regulated and may appear in the serum (W. Schmiegel et al.,Gastroenterology 1990; 99:1421-1430). Serum levels are also raised inseveral gastrointestinal diseases (Satomura et al., J Gastroenterol1995, 30:643-650). The function of PSP/reg is still highly debated, butit is generally assumed that it is involved in promoting cellproliferation during regenerative processes (Y. Kinoshita et al., J.Gastroenterol 2004, 39:507-513). Although this protein is a secretionproduct, its expression is not induced by diet alone. In traumapatients, it has been shown that PSP/reg is up-regulated in blood aftertrauma, and that the PSP/reg level is related to the severity ofinflammation. In particular, it is highly increased in patients duringsepsis (M. Keel et al., Crit Care Med. 2009 37(5):1642-8).

SUMMARY OF THE INVENTION

The present invention relates to a method of prediction and/or diagnosisof infections or inflammatory diseases of the airways and lungs inhuman, in particular for the prediction of the development and theseverity and prognosis of low respiratory tract infection orinflammatory disease including COPD or VAP, wherein the level of PSP/regis determined in a body fluid sample, and a high level is indicative ofthe development and the severity of lower respiratory tract infectionsat early stages of the disease. In addition, in the present invention,high levels of PSP/reg are predictive of survival in ventilator-acquiredpneumonia and correlate with clinical score. Also, high levels ofPSP/reg allow detecting COPD patients with positive sputum microbiology.

DESCRIPTION OF THE FIGURES

FIG. 1: Correlation of SOFA and ln(PSP/reg serum levels) at VAP-onset(p<0.001) in 101 VAP patients. PSP/reg was logarithmized for graphicaldemonstration. Regression line (dashed line).

FIG. 2: Kinetics of PSP/reg serum levels in the groups of survivors (S;n=82) and non-survivors (NS; n=19) in a total of 101 patients with VAPduring the first 7 days (d) after VAP-onset. Boxes represent theinterquartile range (IQR), whiskers include 1.5 times the interquartilerange. For clear graphical presentation outliers are not displayed.Mortality was measured at day 28.

FIG. 3: PSP/reg in receiver operating characteristic (ROC) analysis: atday 0 to predict survival (A), at day 7 to predict death (B). Circleshighlight the most accurate cut-offs. Using the 24 ng/ml cut-off atVAP-onset reveals a sensitivity of 36% and a specificity of 100% topredict survival (AUC: 0.69; FIG. 3A). 177 ng/ml at day 7 was the bestthreshold to predict death with a sensitivity of 54% and a specificityof 90% (AUC: 0.76; FIG. 3B).

FIG. 4: PSP/reg serum levels at exacerbation of COPD according to sputumbacteriology. Patients with positive sputum bacteriology (P; n=62) hadsignificantly higher PSP/reg serum levels as compared to patients withnegative sputum bacteriology (N; n=43) (26.7 ng/ml [19.3-38.8] versus20.8 [15.3-27.2], p=0.005).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of prognosis and/or diagnosisof infections or inflammatory diseases of the airways and lungs inhumans wherein the level of pancreatic stone protein/regeneratingprotein (PSP/reg) is determined in a body fluid sample, and a high levelis indicative of the development and the severity of the disease. In afirst aspect, the invention relates to an ex-vivo method for detectingand/or diagnosing infections or inflammatory diseases of the airways andlungs in humans. In particular, the present method according to theinvention allows the prediction of the severity and the prognosis ofinfections and/or inflammatory diseases of the low respiratory tractincluding COPD and VAP. The methods according to the present inventioncomprise determining the level of PSP/reg in an isolated body fluidsample, e.g. serum, wherein said level is indicative of the severity oflower respiratory tract infections and/or inflammations at early stagesof the disease and of the complications and risk of mortality linked tosaid infections and/or inflammations.

As defined herewith, “patient” refers to any mammalian animal includinghuman, dog, cat, cattle, goat, pig, swine, sheep and monkey. Patientsare preferably humans.

As defined herewith, “lower respiratory tract infection” refers to aninfection in the airways or lungs caused by bacteria, viruses, fungi orparasites. It includes, for example, acute and chronic bronchitis,community-acquired pneumonia, which can be caused, among others, byStreptococcus pneumoniae, atypical bacteria, gram-negative bacteria, orHaemophilus influenza, hospital-acquired pneumonia such as ventilatorassociated pneumonia (VAP), chronic obstructive pulmonary disease(COPD), acute exacerbation chronic obstructive pulmonary disease(AECOPD), bronchiectasis including cystic fibrosis and interstitial lungdisease.

As defined herewith, “ventilator associated pneumonia (VAP)” is apneumonia that develops 48 hours or longer after mechanical ventilationand that is characterized by an invasion of the lower respiratory tractand lung parenchyma by microorganisms. VAP is a potentially seriousmedical condition than can lead to sepsis through the development of asystemic infection that affects a number of organs and tissues, oraffects the body as a whole.

As defined herewith, “chronic obstructive pulmonary disease (COPD)” ischaracterized by a severe obstruction of the airflows that cause anabnormal inflammatory response in the lung. Generally, patients sufferexacerbations that can be triggered by infection or air pollution, andthat lead to AECOPD (acute exacerbation COPD) a critical conditionrequiring hospitalization. COPD can be classified into 4 stagescorrelated with severity (minor, moderated, severe, and very severe)(Gomez and Rodriguez-Roisin, Curr. Opin. Pulm. Med. 2002: 8(2): 81-86).

As defined herewith, “acute exacerbation COPD (AECOPD)” is a suddenworsening of COPD symptoms, like shortness of breath, quantity and colorof phlegm, that typically lasts for several days. It may be triggered byan infection with bacteria or viruses or by environmental pollutants.

In VAP, the infection occurs consequently to surgery, in particularconsequently to an intubation for mechanical ventilation given by meansof an endotracheal tube or tracheotomy. In COPD, the infection can alsooccur consequently to an obstruction of the airways in the lung. Theinfection can originate from the respiratory tract.

As defined herewith, PSP/reg refers to human pancreatic stone protein,also called regenerating gene (REG) I protein or lithostatine orpancreatic thread protein (Gross al., J. Clin. Invest., 1985: 76;2115-2126) and can be the isoform alpha (Uniprot sequence number: P05451also identified herewith as SEQ ID NO. 1) or beta (Uniprot sequencenumber: P48304 also identified herewith as SEQ ID NO. 2).

As defined herewith body sample refers to any sample that is obtainedfrom the patient's body. Body sample includes body fluid samples andextracts from solid tissue or from fecal matter. Body fluid samplesinclude, for instance, samples of whole blood, serum, plasma, urine,sputum, cerebrospinal fluid, tear fluid, sweat, or milk.

One aspect of the invention relates to a method of prognosis and/ordiagnosis of infections or inflammatory diseases of the airways andlungs in a patient, preferably a human, wherein the level of pancreaticstone protein/regenerating protein (PSP/reg) is determined in a bodyfluid sample or in a solid body tissue, and a high level is indicativeof the development and the severity of the disease.

In a further aspect of the method of the invention, the infection in theairways or lungs is caused by bacteria, viruses, fungi, or parasites,and is selected from the group of low respiratory tract infections,particularly acute and chronic bronchitis, acute exacerbation of COPD,stable COPD, community or hospital-acquired or ventilator associatedpneumonia, sarcoidosis, bronchiectasis, including cystic fibrosis andinterstitial lung disease and asthma.

In another aspect, the present invention is directed to an ex-vivomethod of prognosis and/or diagnosis of infections or inflammatorydiseases of the airways and lungs in a patient, or an ex-vivo method fordetecting the development of infections or inflammatory diseases of theairways and lungs in a patient, comprising determining the level ofpancreatic stone protein/regenerating protein (PSP/reg) in a body fluidsample from said patient. The patient is preferably a human.

In another aspect of the method of the invention, the PSP/reg proteinhas the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 2.

In a preferred aspect, the method of the invention is for the diagnosisand prognosis of lower respiratory tract infection, preferablypneumonia, preferably VAP or COPD and AECOPD.

According to a further aspect, the method of the invention is an invitro diagnostic method.

In a still further aspect, the present invention relates to an ex-vivomethod of prognosis and/or diagnosis of infections or inflammatorydiseases of the airways and lungs in a patient, or to an ex-vivo methodfor detecting the development of infections or inflammatory diseases ofthe airways and lungs in a patient, comprising:

-   -   a) providing a body fluid sample from said patient;    -   b) determining the level of PSP/reg in said sample;    -   c) comparing the level of PSP/reg determined in step b) with a        reference value;        wherein a higher level of PSP/reg determined in step b),        compared to the reference value, is indicative of the severity        of the infection and/or inflammatory disease and predictive of        the outcome.

According to another aspect of the invention, the method of theinvention is an in vitro diagnostic method.

In a specific aspect of the invention, the reference value is the levelof PSP/reg measured in a body fluid sample from a patient without knownor suspected infection.

In another aspect of the invention, the reference value is about 10ng/ml.

In a further aspect of the invention, the reference value is 25 ng/ml.In a still further aspect of the invention, the reference value is 180ng/ml, more preferably 200 ng/ml.

In another specific aspect of the invention, the body fluid sample isserum or plasma. Other body fluids than serum and plasma useful fordetermination of PSP/reg levels are e.g. whole blood, urine, sputum,cerebrospinal fluid, tear fluid, sweat, milk, or extracts from solidtissue or from fecal matter.

In COPD patients, the PSP/reg level indicative for bacterialexacerbation in blood serum and plasma is of, or about, 25 ng/ml or morethan 25 ng/ml at exacerbation. Hence, more specifically, the inventionrelates to a method for detecting the presence of positive bacterialsputum as a cause of the exacerbation, wherein the level of PSP/reg isdetermined preferably in serum or plasma, and a level of, or about, 25ng/ml or more, is indicative of positive sputum microbiology.Preferably, a PSP/reg level in serum or plasma that is equal to, orhigher than, about 30 ng/ml is indicative of bacterial exacerbation inCOPD patients.

Also, preferably, the PSP/reg level is determined at exacerbation ofCOPD, or on the day of admission to the hospital following exacerbation.

In VAP patients, the PSP/reg level indicative for severe complicationsand patient outcome, in blood serum and plasma, is of or about 180 ng/mlor more than 180 ng/ml at VAP onset, or at day 2, 3, 4, 5, 6, 7, after aVAP onset that occurred at day 0. Hence, more specifically, theinvention relates to a method to identify individuals with a particulargood and poor outcome and hence to predict severity of infection,wherein the level of PSP/reg is determined in serum or plasma. A PSP/reglevel in serum or plasma of, or below, 24 ng/ml is associated with agood chance of survival, whereas a PSP/reg level of, or higher than, 180ng/ml is indicative of a very poor outcome and high risk of mortality.Preferably, a PSP/reg level of about, or higher than, 200 ng/ml or morepreferably higher than 250 ng/ml is indicative of severe complicationsand poor outcome. Preferably, the PSP/reg level is determined atexacerbation or at day 2, 3, 4, 5, 6, 7 after VAP onset.

In a preferred aspect of the ex-vivo method of the invention, the levelof PSP/reg determined in step b) is determined on the day of VAP onsetor on day 2, 3, 4, 5, 6, 7 after onset as compared to a reference valueof, or about, 10 ng/ml.

In an alternative preferred aspect of the ex-vivo method of theinvention, the level of PSP/reg determined in step b) is determined onthe day of, or on the day of admission to the hospital for, exacerbationof COPD as compared to a reference value of, or about, 10 ng/ml.

Any known method may be used for the determination of the level ofPSP/reg in body fluids. Methods considered are e.g. Enzyme-linkedimmunosorbent assay (ELISA), Radioimmunoassay (RIA), Enzymoimmunoassay(EIA), mass spectrometry, or microarray analysis. Such methods when usedfor the detection of the development of local or systemic infection, inparticular of the detection of the development of sepsis, are a furtherobject of the invention.

A preferred method for the determination of PSP/reg in human bodyfluids, e.g. serum or plasma, is an ELISA. In one embodiment of theinvention, the PSP/reg ELISA consists of a sandwich array: conventionalmicrotiter plates are coated with one type of antibody (“first”antibody”) directed against PSP/reg. The plates are then blocked and thesample or standard is loaded. After the incubation, a different type ofantibody (“second” antibody) against PSP/reg conjugated with a suitablelabel, e.g. an enzyme for chromogenic detection is applied. Finally theplate is developed with a substrate for the label in order to detect andquantify the label, being a measure for the presence and amount ofPSP/reg. If the label is an enzyme for chromogenic detection, thesubstrate is a colour-generating substrate of the conjugated enzyme. Thecolour reaction is then detected in a microplate reader and compared tostandards.

Suitable pairs of antibodies (“first” and “second” antibody) are anycombination of guinea pig, rat, mouse, rabbit, goat, chicken, donkey orhorse. Preferred are monoclonal antibodies, but it is also possible touse polyclonal antibodies or antibody fragments. Suitable labels arechromogenic labels, i.e. enzymes which can be used to convert asubstrate to a detectable coloured or fluorescent compound,spectroscopic labels, e.g. fluorescent labels or labels presenting avisible colour, affinity labels which may be developed by a furthercompound specific for the label and allowing easy detection andquantification, or any other label used in standard ELISA.

Other preferred methods of PSP/reg detection are radioimmunoassay orcompetitive immunoassay using a single antibody and chemiluminescencedetection on automated commercial analytical robots. Microparticleenhanced fluorescence, fluorescence polarized methodologies, or massspectrometry may also be used. Detection devices, e.g. microarrays, areuseful components as readout systems for PSP/reg.

PSP/reg is a protein that can be cloned from pancreatic mRNA andsubcloned into a yeast expression vector. The protein can then beexpressed under the control of Alcohol Dehydrogenase (ADH) promoter. Asuitable expression medium may comprise methanol to induce and maintainthe secretion of PSP/reg. PSP/reg is preferably purified usingSP-Sepharose-cellulose by a pH and salt gradient. Such purified PSP/regis used to prepare standard solutions for comparison with PSP/reg levelsin body fluids. Polyclonal antibodies against the protein may beobtained from mice, rats, rabbits, goats, chicken, donkey, horses andguinea pigs or other suitable animals using standard methods.

The invention further relates to a kit of parts for the determination ofPSP/reg for diagnosis/prediction of infection and/or inflammation of theairways and lungs comprising, for example, apparatus, reagents andstandard solutions of PSP/reg. Apparatus considered are e.g. microtiterplates for ELISA, pre-coated ELISA plates, and plate covers. Reagentsare those reagents particularly developed and designed for the detectionof PSP/reg. Standard solutions of PSP/reg preferably contain PSP/regsynthesized according to the directions hereinbelow. The kit of partsmay contain further hardware, such as pipettes, solutions such asbuffers, blocking solutions and the like, filters, color tables anddirections for use.

In another aspect, the invention relates to a kit for detectingbacterial exacerbation in COPD patients according to a method of theinvention comprising at least one antibody directed against PSP/reg andreagents.

In another aspect, the invention relates to a kit for predicting theseverity of an infection in a VAP patient and patient outcome accordingto a method of the invention comprising at least one antibody directedagainst PSP/reg and reagents.

In a further aspect, the invention relates to a kit for detecting thelevel of pancreatic stone protein (PSP/reg) in a body fluid samplecomprising at least one antibody directed against PSP/reg and reagents.

In a further aspect, the invention relates to the use of a kit accordingto the invention for detecting the development of a local or systemicinfection of the airways and lungs in a patient, notably the developmentof sepsis, for example, in a method according to the invention.

Antibodies directed against PSP/reg can be produced by standard methodsin the field including polyclonal antibody production, monoclonalantibody production. Preferably, the antibodies are directed against aPSP/reg protein having the amino acid sequence of SEQ ID NO:1 or SEQ IDNO: 2.

It is shown that the level of PSP/reg is highly increased in patientsduring VAP with increased risk of mortality and in the case of bacterialexacerbation of COPD. The detection and quantification of serum PSP/regis accomplished e.g. by a sandwich ELISA. Normal serum values are about10 ng/ml. In VAP, the serum values correlate with the severity of VAPand may reach over 200 ng/ml. These values allow predicting u) whether aVAP patient has a good or a poor chance of survival. In COPD, the serumPSP/reg values correlate with the presence of positive bacterial sputumand are above 25 ng/ml. In both VAP and COPD, respective PSP/reg valuesallow predicting the need for intensive treatment including costlyantibiotic treatment and prolonged stay in the intensive care unit.Compared to commercially available diagnostic assays, the PSP/reg ELISAis a reliable assay to predict severity and outcome in VAP patient, andto diagnose bacterial exacerbation of COPD.

The method of the invention and/or the kit according to the inventionare useful for sorting the patients according to groups of risksregarding severity and outcome of the infection or inflammatory disease.

In VAP patients, a PSP/reg level, preferably from a sample obtained fromthe serum or plasma of a VAP patient below or equal to 24 ng/ml ispredictive of a good chance of survival, whereas a PSP/reg level higherthan or equal to 150 ng/ml, preferably higher than180 ng/ml, morepreferably higher than or equal to 200 ng/ml, more preferably higherthan or equal to 250 ng/ml is associated with a high risk of mortalitythat could occur within a variable period comprised between 1 day to 3years, for instance within 28 days after hospitalization.

As a consequence, regular and intensive follow-up and care, possiblyincluding antibiotic treatment and prolonged stay, should be applied toVAP patients belonging to the group(s) of high risk(s).

In COPD patients, a PSP/reg level, preferably from a sample obtainedfrom the serum or plasma of a COPD patient, below 25 ng/ml, morepreferably below 20 mg/ml, is associated with a negative bacterialsputum, whereas a PSP/reg level equal or higher than 25 ng/ml,preferably equal or higher than 30 ng/ml, more preferably equal orhigher than 50 ng/ml, is indicative of bacterial exacerbation. Regardingmortality of COPD patients, a PSP/reg level, preferably from a sampleobtained from the serum or plasma of a COPD patient, below 18 ng/ml,preferably below 15 ng/ml, is associated with a low risk of mortality,whereas a PSP/reg level higher than 30 ng/ml, preferably higher than 34ng/ml or more preferably higher than 35 ng/ml is associated with a highrisk of mortality that could occur within a variable period comprisedbetween 1 day to 3 years, for instance within 2 years afterhospitalization.

As a consequence, regular and intensive follow-up and care, possiblyincluding antibiotic treatment and prolonged stay, should be applied toCOPD patients belonging to the group(s) of high risk(s).

EXAMPLES Example 1 PSP/reg is a Suitable Marker to Assess DiseaseSeverity and Stratify Risk in VAP

It has been investigated whether PSP/reg is predictive for survival inventilator associated pneumonia.

Test Patients

The study included 101 patients (mean age, 56 years) with clinicallydiagnosed VAP. Patients deceased within 28 days were classified asnon-survivors (NS). PSP/reg levels and sequential organ failureassessment (SOFA) scores were obtained on the day of VAP-onset and for10 consecutive days. Detailed baseline characteristics for survivors andnon-survivors are summarized in Table 1. Despite high antibioticpre-treatment within 14 days prior to study inclusion (75%), respiratoryspecimens identified a causative organism in 74 patients (76%). The mostfrequent pathogens were Staphylococcus aureus (30%), Pseudomonasaeruginosa (25%) and Klebsiella species (13%). Appropriate initialantibiotic therapy, defined as a regimen combining an aminoglycoside ora fluoroquinolone plus a betalactam or an antipseudomonal carbapenem wasapplied in 86% of cases. Twenty patients (20%) died during the studyperiod. Deaths were due to traumatic brain injury/subarachnoidhemorrhage (n=8), respiratory failure/Acute Respiratory Distress Syndrom(ARDS) (n=5), septic shock (n=3), cardiogenic shock (n=2), multiorganfailure (n=1) and acute liver failure (n=1). In 7 patients PSP/regvalues at VAP-onset were missing.

TABLE 1 Demographics of 101 VAP patients at study inclusion TotalSurvivors Non-survivors Characteristics n = 101 n = 81 n = 20 p-valueGender (male) (%) 74 (74%) 61 (76%) 13 (65%) 0.459 Age (in years)(range) 57 [43-70] 55 [42-68] 67 [52-75] 0.033 Admission Medical 53(53%) 41 (51%) 12 (60%) 0.615 Elective surgery 4 (4%) 4 (5%) 0 (0%)0.582 Emergency surgery 43 (43%) 35 (43%) 8 (40%) 0.994 From home 61(62%) 51 (65%) 10 (47%) 0.291 From hospital 20 (20%) 15 (19%) 5 (24%)0.517 From other ICU 18 (18%) 12 (15%) 6 (29%) 0.114 Duration ofmechanical 6 [3.5-9] 6 [3-9] 5.5 [4-9.8] 0.801 ventilation before VAP(in days) (range) Antibiotics within 14 days 76 (75%) 59 (73%) 17 (85%)0.387 before VAP-onset Microbiology Positive microbiological 74 (76%) 58(73%) 16 (89%) 0.226 cultures (EA, BAL, PSB) Positive blood cultures 34(34%) 29 (36%) 5 (25%) 0.515 Clinical scores at VAP-onset SAPS II 40.5[32.3-51] 38.0 [31-47.0] 48 [42.0-55] 0.002 ODIN-score 2 [1-3] 2 [1-2] 3[1-4] 0.050 SOFA-score 7.0 [6.0-9.8] 6.0 [5-9] 9.0 [7.0-14] 0.002 Valuesare Means (): percentage values []: range EA: endotracheal aspiratesBAL: bronchoalveolar lavage PSB: protected specimen brush SAPS II:simplified acute physiologic score II ODIN: organ dysfunction and/orinfection SOFA: sepsis-related organ failure assessment

Baseline Assessment and Follow-Up

At time of enrollment the following information was recorded from eachsubject: age, gender, preexisting comorbidities, primary reason forinitiating mechanical ventilation, duration of prior mechanicalventilation, antibiotic use within 14 days of VAP-onset, bodytemperature, heart rate, mean arterial pressure (MAP), oxygensaturation, ratio of partial pressure of arterial oxygen to the fractionof inspired oxygen (PaO₂/FIO₂), leukocyte count (WBC) and PSP/reg serumlevels. The following indices were calculated: simplified acutephysiologic score II (SAPS II), sequential related organ failureassessment (SOFA) score, organ dysfunction and/or infection (ODIN)score. During the 28-day follow-up period the following information wasrecorded: body temperature, heart rate, MAP, oxygen saturation,PaO2/FIO2, WBC, SOFA and ODIN; mechanical ventilation status andantibiotic use and survival throughout the 28-day study period. SerumPSP/reg levels were determined on VAP-onset and for 6 consecutive daysafter VAP diagnosis.

Diagnostic Criteria

Diagnosis of VAP was established on a clinical approach according to theAmerican Thoracic Society Guidelines (Guidelines for the management ofadults with hospital-acquired, ventilator-associated, andhealthcare-associated pneumonia. Am J Respir Crit Care Med 2005;171:388-416). It was defined as a new or progressive infiltrate on chestradiography associated with at least two of the following: purulenttracheal secretions, fever (body temperature>38° C./100.4° F.),leukocytosis/-penia (leukocyte count>11 000/μL or <3 000/μL). VAPpatients were eligible for the study if they were intubated formechanical ventilation for at least 48 hours and older than 18 years.Patients were excluded if they were pregnant, had receivedimmunosuppressants or long-term corticosteroid therapy (above 0.5 mg/kgper day for longer than 1 month), were immunosuppressed or had acoexisting extrapulmonary infection diagnosed in the first three daysrequiring antibiotic therapy for more than three days.

Outcome Assessment

Follow-up was for 28 days or until death. Patients who died within 28days after VAP-onset were classified as non-survivors (NS), all otherswere classified as survivors (S). No patient was lost to follow-up.

PSP/reg Correlated with SOFA

Median [IQR] PSP/reg on VAP-onset was 38.6 ng/ml [22.2-179.0]. There wasno association of PSP/reg with gender, co-morbidities (renal, pulmonary,cardiac, hematological/oncological) and gas exchange (oxygen saturation,PaO₂/FIO₂). PSP/reg correlated with SOFA at VAP-onset (Spearman rankcorrelation coefficient 0.49; p<0.001, FIG. 1) and up to seven days (allp<0.02).

PSP/reg Levels are Predictor of Outcome

PSP/reg concentrations in the serum or plasma were measured byimmunoassay (ELISA method).

PSP/reg levels at VAP-onset were significantly elevated in non-survivors(median [IQR]; 117.0 ng/ml [36.1-295.3], n=19) as compared to survivors(36.3 ng/ml [21.0-124.0], n=75; p=0.011) (FIG. 2). PSP/reg tertiles atVAP-onset differed significantly in survival (log rank p=0.014). Inreceiver operating characteristic analysis, the area under the curve ofPSP/reg for mortality/survival on VAP-onset and after 7 days were 0.69and 0.76 (95% Confidence Interval (CI): 0.57-0.80 and 0.62-0.91),respectively (FIG. 3). PSP/reg below 24 ng/ml at VAP-onset was the mostaccurate threshold for predicting survival. The sensitivity was 36% andspecificity 100% for predicting survival (FIG. 3A). In contrast PSP/regabove 177 ng/ml at day 7 after VAP-onset was the best cut-off to predictdeath (sensitivity: 58% specificity: 91%) (FIG. 3B). Positive andnegative predictive values were 54% and 90%, accordingly. The odds ratiofor patients PSP/reg above 177 ng/ml at day 7 to die until day 28 was13.8 (95% Confidence Interval (CI): 3.3-57.1).

Example 2 PSP/reg is Increased in Acute Exacerbations of COPD withPositive Sputum Culture

It has been investigated whether serum and plasma PSP/reg is increasedin acute exacerbations of COPD and whether PSP/reg detects positivesputum in COPD.

Test Patients

200 patients admitted to hospital for acute exacerbation of COPD wereexamined and were followed up for 2 years. The short and long termfollow-up visits were performed 14-18 days, 6 months and 2 years afterhospital admission, and comprised clinical, laboratory and lung functionassessments. Clinical outcome data were obtained from medical recordsfrom hospital admission and family physicians. Patients were classifiedas clinical success or clinical failure. Clinical failure was defined bythe occurrence of an exacerbation of COPD requiring hospitalisation ordeath of any cause. For survival analysis all patients were classifiedas survivors or non-survivors after two year follow-up. All causemortality was assessed at 6 months and 2 years. For laboratory short-and long-term outcome inflammatory biomarkers (e.g. (PSP/reg),Procalcitonin, C-reactive protein (CRP)) were measured at initialhospital admission and during further course. PSP/reg levels weremeasured in serum and plasma at admission and after 14-18 days.

Study Population

Detailed baseline characteristics of the 200 patients are presented inTable 2. As suggested by the GOLD classification (Gomez andRodriguez-Roisin, Curr. Opin. Pulm. Med. 2002: 8(2): 81-86), COPDseverity as assessed at 14 days was mild in 24 (12%) patients (stage I)and moderate in 41 (21%) (stage II). Most patients had severe or verysevere COPD with stage III in 82 (42%), and stage IV in 50 patients(25%). There were 100 (50%) type I, 44 (22%) type II and 56 (28%) typeIII exacerbations according to the Anthonisen criteria (Anthonisen etal. Ann. Intern. Med. 1987: 106(2): 196-204). Increased sputumproduction and discoloured sputum was present in 139 (69.5%) and 116(58%) patients, respectively. 154 patients (77%) had at least oneexacerbation. At admission, sputum samples for microbiology wereobtained from 113 patients (56.5%). Sputum samples from 8 patients (7%)were from poor quality and excluded from analysis. Sputum cultures grewbacterial pathogens in 63 (56%) of cases. 42 patients (37%) had negativebacterial sputum cultures. 42 patients (68%) with positive sputumspecimens were treated with antibiotics.

TABLE 2 Baseline characteristics of 200 patients presenting with anacute exacerbationof COPD Characteristics N = 200 Gender (male/female)(%) 114/86 (57/43) Age (in years) (range) 70.4 (42-91) Mean Duration ofCOPD (in months) (SD) 125.6 (+/−83.2) Duration AECOPD (in days) (SD) 6.9(+/−10) AECOPD in previous year (at least one) (%) 154 (77) Severity ofCOPD & GOLD Stage (%) I (FEV1% > 80% predicted) 24 (12) II (50% pred. >FEV1% < 80% pred.) 41 (21) III (30% pred. > FEV1% < 50% pred.) 82 (42)IV (FEV1% < 30% pred.) 50 (25) FEV1 predicted (%) (SD) 40 (+/−18.3) FEV1(Liter) (SD) 1.07 (+/−0.52) Chemistry: Leucocyte counts × 10⁹/l 11.1(+/−4.8) C reactive Protein (CRP) mg/dl (SD) 35.1 (+/−46.4)Procalcitonin ng/dl (SD) 0.26 (+/−0.82) Values are absolute numbers (%)or means (standard deviation), when not otherwise indicated, SD =standard deviation; AECOPD denotes acute exacerbation of chronicobstructive lung disease; FEV1 = forced expiratory volume in one second

PSP/reg Serum and Plasma Levels in AECOPD and Recovery

Add a brief description on how the PSP/reg levels were measured, fromwhat kind of sample.

PSP/reg concentrations in the serum or plasma were measured byimmunoassay (ELISA method)

As compared to healthy controls (10.4 [7.5-12.3] ng/ml, n=38), PSP/regwas significantly increased at admission for acute exacerbation of COPD(24.3 [18.4-34.5] ng/ml). However, there was no difference betweenPSP/reg levels at exacerbation and at recovery (p=0.434). PSP/reg levelsdiffered significantly across GOLD stages (p=0.011). There was a goodpositive correlation between PSP serum levels on initial admission andCOPD severity as suggested by the GOLD classification (p=0.016). Medianserum levels of PSP/reg were 20.4 ng/ml [16.9-33.6] for GOLD I, 23.8ng/ml [18.8-32.8] for GOLD II, 26.4 ng/ml [20.6-38.8] for GOLD III and20.8 ng/ml [16.3-29.3] for GOLD IV. PSP/reg levels correlated withC-reactive protein (p=0.031) and procalcitonin (p<0.001).

PSP/reg Serum and Plasma Levels and Positive Sputum Bacteriology

Patients with positive and negative sputum bacterial cultures differedsignificantly in regard of PSP/reg serum values. Patients with positivesputum microbiology had higher levels of PSP/reg (26.7 ng/ml [19.2-38.5]vs. 20.8 ng/ml [15.6-27.2], p=0.008) (FIG. 4).

PSP/reg and Length of Hospital-Stay and 2 Year Mortality

PSP/reg levels at exacerbation correlated significantly with the lengthof hospital stay (r=0.231; p=0.001). Patients with PSP/reg levels <25thpercentile (<18.36 ng/ml) had a mean hospital stay from 6.9 days (±7.62)as compared to 11.34 days (±7.42) in patients presenting withlevels >75th percentile (>33.86 ng/ml) on admission for exacerbation ofCOPD. In a Kaplan-Meier analysis to evaluate the potential of PSP/reglevels to predict short- and long-term mortality at 2-years there was asignificant difference in the cumulative risk of death within the 2years after hospitalisation for patients with lower (<18.36 ng/ml),intermediate (18.36-33.86 ng/ml) and higher PSP/reg levels (>33.86ng/ml), (p<0.001).

1. A ex-vivo method of prognosis and/or diagnosis of infections of theairways or lungs in a patient comprising determining a level ofpancreatic stone protein/regenerating protein (PSP/reg) in a body fluidsample from said patient, and making a prognosis and/or diagnosis ofinfections and/or inflammatory diseases of the airways or lungs in thepatient, and, optionally, treating said infections and/or inflammatorydiseases.
 2. The method of claim 1, comprising: a) providing a bodyfluid sample from said patient; b) determining the level of PSP/reg insaid sample; c) comparing the level of PSP/reg determined in step b)with a reference value; wherein a higher level of PSP/reg determined instep b), compared to the reference value, is indicative of a severity ofsaid infection and predictive of its outcome.
 3. The method of claim 1,wherein said body fluid sample is a serum sample or a plasma sample. 4.The method of claim 1, wherein said patient is a human.
 5. The method ofclaim 1, wherein said PSP/reg protein has an amino acid sequence of SEQID NO:1 or SEQ ID NO:
 2. 6. The method of claim 2, wherein the referencevalue is obtained by determining the level of PSP/reg in a body fluidsample from a patient without infection.
 7. The method of claim 2,wherein the reference value is below 20 ng/ml.
 8. The method of claim 1,wherein the infection of the airways or lungs is selected from the groupof low respiratory tract infections.
 9. The method of claim 2, wherein alevel of PSP/reg equal or higher than the reference value is indicativeof an infection of the airways and lungs in said patient.
 10. The methodof claim 1, wherein a level of PSP/reg equal or higher than 150 ng/ml isindicative of disease severity and predictive of mortality in aventilator-acquired pneumonia (VAP) patient.
 11. The method of claim 1,wherein a level of PSP/reg equal or higher than 25 ng/ml, is indicativeof positive sputum microbiology in a COPD patient.
 12. The method ofclaim 1, wherein the level of PSP/reg in said body fluid sample isdetermined by Enzyme-linked immunosorbent assay (ELISA),Radioimmunoassay (RIA), Enzymoimmunoassay (EIA), mass spectrometry, ormicroarray analysis.
 13. The method of claim 1, wherein the level ofPSP/reg is determined by a sandwich ELISA, wherein microtiter plates arecoated with one type of antibody directed against PSP/reg, the platesare then blocked and the sample or standard is loaded, a second type ofantibody against PSP/reg is applied, a third antibody detecting theparticular type of the second antibody conjugated with a suitable labelis then added, and the label used to quantify the amount of PSP/reg. 14.The method of claim 13, wherein the label in the sandwich ELISA is anenzyme for chromogenic detection.
 15. A kit of parts for determiningPSP/reg for diagnosis/prediction of infection of airways and lungs in apatient comprising apparatus, reagents and standard solutions ofPSP/reg.
 16. The method of claim 7, wherein the reference value is 10ng/ml.
 17. The method of claim 8, wherein the inflammatory disease ofthe airways or lungs is selected from the group of acute bronchitis,acute exacerbation of COPD, community or hospital-acquired or ventilatorassociated pneumonia and interstitial lung disease.
 18. The method ofclaim 10, wherein a level of PSP/reg is higher than 180 ng/ml, or higherthan 200 ng/ml.
 19. The method of claim 19, wherein based on the levelof PSP/reg determined, the patient is grouped with regard to risksregarding severity and outcome of the infection.
 20. The method of claim19, wherein (i) ventilator-acquired pneumonia (VAP) patients are groupedas follows: PSP/reg level below or equal to 24 ng/ml: good chance ofsurvival, PSP/reg level higher than or equal to 150 ng/ml: high risk ofmortality within a variable period between 1 day to 3 years; (ii)chronic obstructive pulmonary disease (COPD) patients are grouped asfollows: PSP/reg level below 18 ng/ml: low risk of mortality, PSP/reglevel above 30 ng/ml: high risk of mortality within a variable periodbetween 1 day to 3 years.